Impact in stability during sequential CDR grafting to construct camelid VHH antibodies against zinc oxide and gold

Impact in stability during sequential CDR grafting to construct camelid VHH antibodies against... Abstract Biomolecules which recognize inorganic materials and metal surfaces gain much attention for creating new type of nanomaterials and sensors. 4F2, a camelid VHH antibody, recognizes ZnO surface and has been applied for sensor applications. 4F2 was constructed sequential complementarity determining region (CDR) replacement on the parental VHH antibody, termed the Construction of Antibody by Integrating Grafting and Evolution Technology; CAnIGET procedure. Here, we evaluate the influence of CDR replacements during 4F2 generation using calorimetric technique. We found that the initial peptide grafting at CDR1 results in the stability reduction and subsequent CDR3 randomize and selection restore the stability during the construction of 4F2. Further examination using anti-gold VHH, AuE32, revealed that the final CDR3 randomize and selection step has little effect in stability while the initial CDR1 grafting reduces the stability as same as the case for 4F2. Our results showing here provide the detailed view of the stability alteration during the CAnIGET procedure. CDR grafting, protein stability, thermal denaturation, VHH antibody Interaction between biomolecules and inorganic materials has gained much attention in recent years (1), because such interactions can be used for several applications, including the construction of new type of conjugate materials (2). Several material recognizing antibody clones, which specifically bind to gold (3, 4), gallium arsenide (5) and zinc oxide (ZnO) (6) surfaces, have been reported. To obtain anti-material antibodies with high affinity and high specificity, we have developed an antibody designing protocol, termed the Construction of Antibody by Integrating Grafting and Evolution Technology; CAnIGET (6). This protocol is based on the sequential complementarity determining region (CDR) loop replacements of a variable domain of camelid heavy chain antibody (VHH). There are three CDR loops in VHH and an inorganic surface recognition peptide sequence was grafted to the CDR1 loop and then the CDR3 loop was randomized. The CDR3 sequence was designed as a αββα repeat where α residues were randomized to Arg or His and β residues were randomized to Arg, Gly, Leu or Val. The resulting VHHs have shown their high affinity and specificity. 4F2 is one of a VHH clone, which obtained by the CAnIGET procedure and recognizes ZnO surface (6), and has been used for several sensor applications (7, 8). We previously reported that 4F2 can recognize Zn2+ and the Zn2+ binding is mainly mediated by the CDR3 loop (9). Although CAnIGET provides the solid framework for the effective generation of antibodies, the detailed evaluation of stability during the CDR replacement have not done yet. In previous study, we have constructed a series of CDR loop variants of 4F2 (6). The 4F2 variants are shown in Fig. 1. The parental VHH antibody is cAbBCII10, which was widely used VHH scaffold for CDR grafting (10–12). In the first step, CDR1 loop was replaced with a ZnO binding peptide (ZnOBP) (13) and the resulting VHH was termed VHHZnOBP1. Then, the CDR3 loop was randomized and high affinity clone was selected by phage display method. We also have constructed a mutant which has CDR3 mutation of 4F2, termed VHHsZnOBP3. (6) The dissociation constants (Kd) towards ZnO particles have been determined to 9 nM (4F2), 176 nM (VHHZnOBP1) and 168 nM (VHHZnOBP1) (6). Fig. 1 View largeDownload slide 4F2 VHH variants used in this study. Three convex loops represent the CDR loops (CDR1, CDR2 and CDR3). Loops with dotted line are the sequence modified loops from wildtype cAbBCII10 during the CAnIGET procedure. Fig. 1 View largeDownload slide 4F2 VHH variants used in this study. Three convex loops represent the CDR loops (CDR1, CDR2 and CDR3). Loops with dotted line are the sequence modified loops from wildtype cAbBCII10 during the CAnIGET procedure. Materials and Methods Protein production BL21(DE3) E. coli cells transformed with the plasmids were grown in LB medium over night at 37 °C. Cells were pelleted and sonicated after suspended with 50 mM phosphate pH 5.0 buffer. The supernatant of the sonicated sample was loaded onto the SP Sepharose cation exchange column (GE healthcare, USA). Then the fractions containing VHH variants were subjected final purification step with Sephacryl S-100 size exclusion chromatography column (GE healthcare, USA). Analytical size-exclusion chromatography Superdex75 10/300 column was used for the analytical size-exclusion chromatography. 50 μL samples were loaded onto the analytical size-exclusion chromatography column equilibrated with 50 mM phosphate pH 7.0 and 150 mM NaCl buffer at room temperature. Differential scanning calorimetry We used a capillary cell type MicroCal VP-capillary differential scanning calorimetry (DSC) (Malvern, USA). All the experiments were done with sample concentration of 1 mg/ml and 60 °C/h scanning rate. Results and Discussion Here, we study the impact in stability during the CDR grafting by using the CDR loop replaced variants (VHHZnOBP1, VHHsZnOBP3 and 4F2). The original constructs of these variants have a FLAG tag at the C-terminus. To avoid any artifacts from the tag, we deleted the FLAG sequence from the expression vectors. Figure 2a shows the SDS-poly acrylamide gel electrophoresis (PAGE) of purified VHH variants. All variants were purified as a single band. Then we performed the size exclusion chromatography. The size exclusion chromatography results suggest all the variants monomeric without the C-terminal FLAG tag (Fig. 2b). Fig. 2 View largeDownload slide (a) SDS-PAGE analysis of purified VHH variants. M: molecular weight maker, 1: cAbBCII10, 2: VHHZnOBP1, 3: VHHsZnOBP3, 4: 4F2. (b) Size-exclusion chromatography of the VHH variants. Fig. 2 View largeDownload slide (a) SDS-PAGE analysis of purified VHH variants. M: molecular weight maker, 1: cAbBCII10, 2: VHHZnOBP1, 3: VHHsZnOBP3, 4: 4F2. (b) Size-exclusion chromatography of the VHH variants. To evaluate the impacts of the CDR grafting, we have determined melting temperatures of the 4F2 variants using a DSC. Figure 3 shows the DSC results measured at pH 7.0 using 100 mM phosphate, 200 mM NaCl buffer. 4F2 shows the melting temperature ™ at 66.9 °C while parental cAbBCII10 shows 78.3 °C. We have previously reported this destabilization behaviour (9) and it indicates that sequential grafting of CDR loops induces the de-stabilization. Because the amino-acid sequence of CDR1 and CDR3 of 4F2 are only the difference from wildtype (Fig. 1), the de-stabilization must be induced by these CDR loop differences. Hence, we then evaluated VHH variants whose only one CDR is replaced (VHHZnOBP1 and VHHsZnOBP3; Fig. 1). VHHZnOBP1 and VHHsZnOBP3 show the Tm values of 61.8 °C and 77.5 °C, respectively (Fig. 3). Fig. 3 View largeDownload slide DSC experiments of 4F2 VHH variants in pH 7.0 buffer. Raw thermogram of the 4F2 variants were subtracted from a buffer baseline. Grey solid line: 4F2, Black solid line: wildtype cAbBCII10 VHH, Dotted line: VHHZnOBP1, Dashed line: VHHsZnOBP3. Sample keys and Tm values are shown on the side of the peaks. Sample concentration of 1 mg/ml and 60 °C/h scanning rate. Fig. 3 View largeDownload slide DSC experiments of 4F2 VHH variants in pH 7.0 buffer. Raw thermogram of the 4F2 variants were subtracted from a buffer baseline. Grey solid line: 4F2, Black solid line: wildtype cAbBCII10 VHH, Dotted line: VHHZnOBP1, Dashed line: VHHsZnOBP3. Sample keys and Tm values are shown on the side of the peaks. Sample concentration of 1 mg/ml and 60 °C/h scanning rate. The most destabilized VHH clone was VHHZnOBP1. VHHsZnOBP3 shows almost equivalent stability with wildtype. It indicates that the CDR1 replacement is the key for the de-stabilization. It should be noted that further optimization of CDR3 on VHHZnOBP1, resulting mutant is 4F2, recovers its stability by 5.1 °C without altering CDR1 sequence. Thus, this stability recovery must be induced by the CDR3 replacement. When the ZnO binding peptide was grafted to CDR1 to construct VHHZnOBP1, no sequence optimization was performed. Thus, the de-stabilization of VHHZnOBP1 may due to a structural incompatibility between the VHH scaffold and the grafting ZnO binding peptide. However, subsequent CDR3 randomization/selection step restored the de-stabilization accompanying affinity improvement. An interesting point is that the CDR3 sequence of 4F2 itself does not improve the VHH stability from the DSC result of VHHsZnOBP3. This observation clearly indicates that the CDR3 sequence of 4F2 co-operatively restore the de-stabilizing effect with CDR1. To evaluate the CDR contribution in stability, we focused on the histidine residues because CDR1 and CDR3 of 4F2 have relatively high content of histidine residues. The CDR1 and CDR3 sequences of 4F2 are EAHVMHKVAPRP and HLGHGLHRVH while wildtype sequences are EYSYSTF (CDR1) and YFMRLPSSHN (CDR3; histidine residues are underlined). We determined the Tm values of 4F2 variants at pH 6.0 (100 mM phosphate, 200 mM NaCl) and pH 5.0 (100 mM sodium acetate, 200 mM NaCl) using DSC. Because the imidazole ring of histidine has a pKa value of 6.0 and below this pKa value, the protonated state of histidine residues can be alternated. Table I shows the Tm vale differences of the 4F2 variants at pH 6.0 and pH 5.0 from the pH 7.0 Tm values. At pH 5.0 conditions, the largest drop of Tm values was observed in all the 4F2 variants. VHHZnOBP1 shows the largest Tm decrease among pH 5.0 data with the value of –4.5 °C while wildtype cAbBCII10 show the smallest decrease with the value of –1.2 °C. 4F2 show second large decrease with the value of –3.7 °C and VHHsZnOBP3 shows the third with –2.1 °C. These data indicate that CDR1 is more susceptible to histidine protonation than CDR3. We initially expected that CDR3 contains four histidine residues and these histidine residues are important for stability. However, the DSC results show that the destabilization of VHHsZnOBP3 at pH 5.0 is relatively small. The Tm data at different pH conditions suggest histidine residues in CDR1 may be more critical for the stability than that in CDR3. Table I. The Tm difference (°C) from pH 7.0 values of 4F2 variants pH Wildtype 4F2 VHHZnOBP1 VHHsZnOBP3 7.0 0.0 °C 0.0 °C 0.0 °C 0.0 °C 6.0 –0.3 °C –0.8 °C –1.2 °C –0.4 °C 5.0 –1.2 °C –3.7 °C –4.5 °C –2.1 °C pH Wildtype 4F2 VHHZnOBP1 VHHsZnOBP3 7.0 0.0 °C 0.0 °C 0.0 °C 0.0 °C 6.0 –0.3 °C –0.8 °C –1.2 °C –0.4 °C 5.0 –1.2 °C –3.7 °C –4.5 °C –2.1 °C Buffers, 100 mM PO4 and 200 mM NaCl (pH 7.0, pH6.0) or 100 mM sodium acetate and 200 mM NaCl (pH 5.0), was used. Table I. The Tm difference (°C) from pH 7.0 values of 4F2 variants pH Wildtype 4F2 VHHZnOBP1 VHHsZnOBP3 7.0 0.0 °C 0.0 °C 0.0 °C 0.0 °C 6.0 –0.3 °C –0.8 °C –1.2 °C –0.4 °C 5.0 –1.2 °C –3.7 °C –4.5 °C –2.1 °C pH Wildtype 4F2 VHHZnOBP1 VHHsZnOBP3 7.0 0.0 °C 0.0 °C 0.0 °C 0.0 °C 6.0 –0.3 °C –0.8 °C –1.2 °C –0.4 °C 5.0 –1.2 °C –3.7 °C –4.5 °C –2.1 °C Buffers, 100 mM PO4 and 200 mM NaCl (pH 7.0, pH6.0) or 100 mM sodium acetate and 200 mM NaCl (pH 5.0), was used. To evaluate the general stability tendency of CAnIGET clones, we further measured the stability of the another CAnIGET VHH clone, AuE32, which binds to gold surface (3). The initial CDR1 grafting of a gold binding peptide (GBP: LKAHLPPSRLPS) resulted in a mutant VHHGBP1 and further CDR3 randomization/selection on VHHGBP1 resulted in the AuE32 VHH clone (CDR3 sequence: RRVRGGHLLR) as shown in Fig. 1. VHHGBP1 and AuE32 show the dissociation constants 3 μM and 150 nM toward gold surface, respectively (3). Both clones were purified using the same purification step of the 4F2 variants (Fig. 4a). We then performed the stability measurements of VHHGBP1 and AuE32 using DSC (Fig. 4b). AuE32 shows the Tm value of 61.0 °C while VHHGBP1 shows the Tm value of 62.3 °C. The CDR1 grafting resulted in the drop of the Tm by 16.0 °C which is similar de-stabilization of VHHZnOBP1 (Tm drop of 16.5 °C). Thus, the initial peptide grafting on CDR1 in the CAnIGET procedure may generally cause the de-stabilization. On the other hand, AuE32 shows a similar Tm value with that of VHHGBP1 with a little Tm drop by 1.3 °C. This stability tendency for the CDR3 randomization/selection step contrasts with that for 4F2, which recovers the stability after the final step. Though the reason for the difference of the stability impact on CDR3 between 4F2 and AuE32 is not clear, one possible reason might be the affinity difference toward material surfaces. In the case of 4F2, the parental VHH (VHHZnOBP1) already has decent affinity toward ZnO surface (Kd: 176 nM), while the parental VHH of AuE32 (VHHGBP1) has relatively low affinity toward gold surface (Kd: 3 μM). Thus, the CDR3 randomization/selection step could be harsher for AuE32 to survive the punning step. We speculate that the stably expressed clone on the phage surface may be relatively easily selected for the case of 4F2 while the less stable clone may be selected for the case of AuE32 because few phage clones with the decent affinity and stability may exist in the library. Fig. 4 View largeDownload slide (a) SDS-PAGE analysis of VHHGBP1 (lane 2) and AuE32 (lane 3). The result for cAbBCII10 and marker are also shown (lane 1and lane M). (b) DSC experiments of AuE32 variants at pH 7.0. Grey solid line: AuE32, Dotted line: VHHGBP1, Black sold line: cAbBCII10. Fig. 4 View largeDownload slide (a) SDS-PAGE analysis of VHHGBP1 (lane 2) and AuE32 (lane 3). The result for cAbBCII10 and marker are also shown (lane 1and lane M). (b) DSC experiments of AuE32 variants at pH 7.0. Grey solid line: AuE32, Dotted line: VHHGBP1, Black sold line: cAbBCII10. It has been reported that the wild-type VHH, cAbBCII-10, is suitable scaffold for CDR grafting (10). The scaffold accepts various types of CDR combinations, including anti-Lysozyme, anti-GFP and anti-RNase A VHH antibodies without significant stability loss. This stable scaffold is important basis for CAnIGET because the initial CDR1 grafting may reduce the stability and the scaffold must resist this initial de-stabilization. In summary, our results showing here demonstrate that the stability of VHH clones are drastically changed during the CAnIGET procedure (6). The initial peptide grafting at CDR1 leads to the de-stabilization of the VHH while subsequent randomization and selection steps on CDR3 show improvement of the stability for the 4F2 case while the case for AuE32 shows a little de-stabilization. To reveal the detailed mechanism of the stability change, further structural characterizations, such as X-ray crystallography, will be needed in the future study. Acknowledgements The authors thank Mrs. Daisuke Takeda and Ryosuke Sasaki for their technical assistances. This study was supported by JSPS KAKENHI Grant Numbers 24000011 and 26670051. Conflict of Interest None declared. References 1 Mirkin C.A. , Taton T.A. ( 2000 ) Materials chemistry: semiconductors meet biology . Nature 405 , 626 – 627 Google Scholar CrossRef Search ADS PubMed 2 Dickerson M.B. , Sandhage K.H. , Naik R.R. ( 2008 ) Protein- and peptide-directed syntheses of inorganic materials . Chem. Rev . 108 , 4935 – 4978 Google Scholar CrossRef Search ADS PubMed 3 Hattori T. , Umetsu M. , Nakanishi T. , Sawai S. , Kikuchi S. , Asano R. , Kumagai I. ( 2012 ) A high-affinity gold-binding camel antibody: antibody engineering for one-pot functionalization of gold nanoparticles as biointerface molecules . Bioconjugate Chem. 23 , 1934 – 1944 Google Scholar CrossRef Search ADS 4 Watanabe H. , Nakanishi T. , Umetsu M. , Kumagai I. ( 2008 ) Human anti-gold antibodies: biofunctionalization of gold nanoparticles and surfaces with anti-gold antibodies . J. Biol. Chem. 283 , 36031 – 36038 Google Scholar CrossRef Search ADS PubMed 5 Artzy Schnirman A. , Zahavi E. , Yeger H. , Rosenfeld R. , Benhar I. , Reiter Y. , Sivan U. ( 2006 ) Antibody molecules discriminate between crystalline facets of a gallium arsenide semiconductor . Nano Lett . 6 , 1870 – 1874 Google Scholar CrossRef Search ADS PubMed 6 Hattori T. , Umetsu M. , Nakanishi T. , Togashi T. , Yokoo N. , Abe H. , Ohara S. , Adschiri T. , Kumagai I. ( 2010 ) High affinity anti-inorganic material antibody generation by integrating graft and evolution technologies: potential of antibodies as biointerface molecules . J. Biol. Chem. 285 , 7784 – 7793 Google Scholar CrossRef Search ADS PubMed 7 Tawa K. , Umetsu M. , Hattori T. , Kumagai I. ( 2011 ) Zinc oxide-coated plasmonic chip modified with a bispecific antibody for sensitive detection of a fluorescent labeled-antigen . Anal. Chem . 83 , 5944 – 5948 Google Scholar CrossRef Search ADS PubMed 8 Tawa K. , Umetsu M. , Nakazawa H. , Hattori T. , Kumagai I. ( 2013 ) Application of 300× enhanced fluorescence on a plasmonic chip modified with a bispecific antibody to a sensitive immunosensor . ACS Appl. Mater. Interfaces . 5 , 8628 – 8632 Google Scholar CrossRef Search ADS PubMed 9 Sasaki R. , Kitazawa S. , Kitahara R. , Nakazawa H. , Tanaka Y. , Kumagai I. , Umetsu M. , Makabe K. ( 2015 ) Zinc ion-binding activity of an anti-ZnO VHH antibody, 4F2 . Chem. Lett . 44 , 1309 – 1311 Google Scholar CrossRef Search ADS 10 Saerens D. , Pellis M. , Loris R. , Pardon E. , Dumoulin M. , Matagne A. , Wyns L. , Muyldermans S. , Conrath K. ( 2005 ) Identification of a universal VHH framework to graft non-canonical antigen-binding loops of camel single-domain antibodies . J. Mol. Biol . 352 , 597 – 607 Google Scholar CrossRef Search ADS PubMed 11 Conrath K.E. , Lauwereys M. , Galleni M. , Matagne A. , Frère J.-M. , Kinne J. , Wyns L. , Muyldermans S. ( 2001 ) β-Lactamase inhibitors derived from single-domain antibody fragments elicited in the camelidae . Antimicrob. Agents Chemotherapy . 45 , 2807 – 2812 Google Scholar CrossRef Search ADS 12 Vincke C. , Loris R. , Saerens D. , Martinez-Rodriguez S. , Muyldermans S. , Conrath K. ( 2009 ) General strategy to humanize a camelid single-domain antibody and identification of a universal humanized nanobody scaffold . J. Biol. Chem . 284 , 3273 – 3284 Google Scholar CrossRef Search ADS PubMed 13 Umetsu M. , Mizuta M. , Tsumoto K. , Ohara S. , Takami S. , Watanabe H. , Kumagai I. , Adschiri T. ( 2005 ) Bioassisted room-temperature immobilization and mineralization of zinc oxide—the structural ordering of ZnO nanoparticles into a flower-type morphology . Adv. Mater. 17 , 2571 – 2575 Google Scholar CrossRef Search ADS Abbreviations Abbreviations CAnIGET construction of antibody by integrating grafting and evolution technology CDR complementarity determining region DSC differential scanning calorimetry SDS-PAGE sodium dodecyl sulfate poly acrylamide gel electrophoresis VHH VH of heavy chain antibody © The Author(s) 2018. Published by Oxford University Press on behalf of the Japanese Biochemical Society. All rights reserved This article is published and distributed under the terms of the Oxford University Press, Standard Journals Publication Model (https://academic.oup.com/journals/pages/about_us/legal/notices) http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png The Journal of Biochemistry Oxford University Press

Impact in stability during sequential CDR grafting to construct camelid VHH antibodies against zinc oxide and gold

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Oxford University Press
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© The Author(s) 2018. Published by Oxford University Press on behalf of the Japanese Biochemical Society. All rights reserved
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0021-924X
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Abstract

Abstract Biomolecules which recognize inorganic materials and metal surfaces gain much attention for creating new type of nanomaterials and sensors. 4F2, a camelid VHH antibody, recognizes ZnO surface and has been applied for sensor applications. 4F2 was constructed sequential complementarity determining region (CDR) replacement on the parental VHH antibody, termed the Construction of Antibody by Integrating Grafting and Evolution Technology; CAnIGET procedure. Here, we evaluate the influence of CDR replacements during 4F2 generation using calorimetric technique. We found that the initial peptide grafting at CDR1 results in the stability reduction and subsequent CDR3 randomize and selection restore the stability during the construction of 4F2. Further examination using anti-gold VHH, AuE32, revealed that the final CDR3 randomize and selection step has little effect in stability while the initial CDR1 grafting reduces the stability as same as the case for 4F2. Our results showing here provide the detailed view of the stability alteration during the CAnIGET procedure. CDR grafting, protein stability, thermal denaturation, VHH antibody Interaction between biomolecules and inorganic materials has gained much attention in recent years (1), because such interactions can be used for several applications, including the construction of new type of conjugate materials (2). Several material recognizing antibody clones, which specifically bind to gold (3, 4), gallium arsenide (5) and zinc oxide (ZnO) (6) surfaces, have been reported. To obtain anti-material antibodies with high affinity and high specificity, we have developed an antibody designing protocol, termed the Construction of Antibody by Integrating Grafting and Evolution Technology; CAnIGET (6). This protocol is based on the sequential complementarity determining region (CDR) loop replacements of a variable domain of camelid heavy chain antibody (VHH). There are three CDR loops in VHH and an inorganic surface recognition peptide sequence was grafted to the CDR1 loop and then the CDR3 loop was randomized. The CDR3 sequence was designed as a αββα repeat where α residues were randomized to Arg or His and β residues were randomized to Arg, Gly, Leu or Val. The resulting VHHs have shown their high affinity and specificity. 4F2 is one of a VHH clone, which obtained by the CAnIGET procedure and recognizes ZnO surface (6), and has been used for several sensor applications (7, 8). We previously reported that 4F2 can recognize Zn2+ and the Zn2+ binding is mainly mediated by the CDR3 loop (9). Although CAnIGET provides the solid framework for the effective generation of antibodies, the detailed evaluation of stability during the CDR replacement have not done yet. In previous study, we have constructed a series of CDR loop variants of 4F2 (6). The 4F2 variants are shown in Fig. 1. The parental VHH antibody is cAbBCII10, which was widely used VHH scaffold for CDR grafting (10–12). In the first step, CDR1 loop was replaced with a ZnO binding peptide (ZnOBP) (13) and the resulting VHH was termed VHHZnOBP1. Then, the CDR3 loop was randomized and high affinity clone was selected by phage display method. We also have constructed a mutant which has CDR3 mutation of 4F2, termed VHHsZnOBP3. (6) The dissociation constants (Kd) towards ZnO particles have been determined to 9 nM (4F2), 176 nM (VHHZnOBP1) and 168 nM (VHHZnOBP1) (6). Fig. 1 View largeDownload slide 4F2 VHH variants used in this study. Three convex loops represent the CDR loops (CDR1, CDR2 and CDR3). Loops with dotted line are the sequence modified loops from wildtype cAbBCII10 during the CAnIGET procedure. Fig. 1 View largeDownload slide 4F2 VHH variants used in this study. Three convex loops represent the CDR loops (CDR1, CDR2 and CDR3). Loops with dotted line are the sequence modified loops from wildtype cAbBCII10 during the CAnIGET procedure. Materials and Methods Protein production BL21(DE3) E. coli cells transformed with the plasmids were grown in LB medium over night at 37 °C. Cells were pelleted and sonicated after suspended with 50 mM phosphate pH 5.0 buffer. The supernatant of the sonicated sample was loaded onto the SP Sepharose cation exchange column (GE healthcare, USA). Then the fractions containing VHH variants were subjected final purification step with Sephacryl S-100 size exclusion chromatography column (GE healthcare, USA). Analytical size-exclusion chromatography Superdex75 10/300 column was used for the analytical size-exclusion chromatography. 50 μL samples were loaded onto the analytical size-exclusion chromatography column equilibrated with 50 mM phosphate pH 7.0 and 150 mM NaCl buffer at room temperature. Differential scanning calorimetry We used a capillary cell type MicroCal VP-capillary differential scanning calorimetry (DSC) (Malvern, USA). All the experiments were done with sample concentration of 1 mg/ml and 60 °C/h scanning rate. Results and Discussion Here, we study the impact in stability during the CDR grafting by using the CDR loop replaced variants (VHHZnOBP1, VHHsZnOBP3 and 4F2). The original constructs of these variants have a FLAG tag at the C-terminus. To avoid any artifacts from the tag, we deleted the FLAG sequence from the expression vectors. Figure 2a shows the SDS-poly acrylamide gel electrophoresis (PAGE) of purified VHH variants. All variants were purified as a single band. Then we performed the size exclusion chromatography. The size exclusion chromatography results suggest all the variants monomeric without the C-terminal FLAG tag (Fig. 2b). Fig. 2 View largeDownload slide (a) SDS-PAGE analysis of purified VHH variants. M: molecular weight maker, 1: cAbBCII10, 2: VHHZnOBP1, 3: VHHsZnOBP3, 4: 4F2. (b) Size-exclusion chromatography of the VHH variants. Fig. 2 View largeDownload slide (a) SDS-PAGE analysis of purified VHH variants. M: molecular weight maker, 1: cAbBCII10, 2: VHHZnOBP1, 3: VHHsZnOBP3, 4: 4F2. (b) Size-exclusion chromatography of the VHH variants. To evaluate the impacts of the CDR grafting, we have determined melting temperatures of the 4F2 variants using a DSC. Figure 3 shows the DSC results measured at pH 7.0 using 100 mM phosphate, 200 mM NaCl buffer. 4F2 shows the melting temperature ™ at 66.9 °C while parental cAbBCII10 shows 78.3 °C. We have previously reported this destabilization behaviour (9) and it indicates that sequential grafting of CDR loops induces the de-stabilization. Because the amino-acid sequence of CDR1 and CDR3 of 4F2 are only the difference from wildtype (Fig. 1), the de-stabilization must be induced by these CDR loop differences. Hence, we then evaluated VHH variants whose only one CDR is replaced (VHHZnOBP1 and VHHsZnOBP3; Fig. 1). VHHZnOBP1 and VHHsZnOBP3 show the Tm values of 61.8 °C and 77.5 °C, respectively (Fig. 3). Fig. 3 View largeDownload slide DSC experiments of 4F2 VHH variants in pH 7.0 buffer. Raw thermogram of the 4F2 variants were subtracted from a buffer baseline. Grey solid line: 4F2, Black solid line: wildtype cAbBCII10 VHH, Dotted line: VHHZnOBP1, Dashed line: VHHsZnOBP3. Sample keys and Tm values are shown on the side of the peaks. Sample concentration of 1 mg/ml and 60 °C/h scanning rate. Fig. 3 View largeDownload slide DSC experiments of 4F2 VHH variants in pH 7.0 buffer. Raw thermogram of the 4F2 variants were subtracted from a buffer baseline. Grey solid line: 4F2, Black solid line: wildtype cAbBCII10 VHH, Dotted line: VHHZnOBP1, Dashed line: VHHsZnOBP3. Sample keys and Tm values are shown on the side of the peaks. Sample concentration of 1 mg/ml and 60 °C/h scanning rate. The most destabilized VHH clone was VHHZnOBP1. VHHsZnOBP3 shows almost equivalent stability with wildtype. It indicates that the CDR1 replacement is the key for the de-stabilization. It should be noted that further optimization of CDR3 on VHHZnOBP1, resulting mutant is 4F2, recovers its stability by 5.1 °C without altering CDR1 sequence. Thus, this stability recovery must be induced by the CDR3 replacement. When the ZnO binding peptide was grafted to CDR1 to construct VHHZnOBP1, no sequence optimization was performed. Thus, the de-stabilization of VHHZnOBP1 may due to a structural incompatibility between the VHH scaffold and the grafting ZnO binding peptide. However, subsequent CDR3 randomization/selection step restored the de-stabilization accompanying affinity improvement. An interesting point is that the CDR3 sequence of 4F2 itself does not improve the VHH stability from the DSC result of VHHsZnOBP3. This observation clearly indicates that the CDR3 sequence of 4F2 co-operatively restore the de-stabilizing effect with CDR1. To evaluate the CDR contribution in stability, we focused on the histidine residues because CDR1 and CDR3 of 4F2 have relatively high content of histidine residues. The CDR1 and CDR3 sequences of 4F2 are EAHVMHKVAPRP and HLGHGLHRVH while wildtype sequences are EYSYSTF (CDR1) and YFMRLPSSHN (CDR3; histidine residues are underlined). We determined the Tm values of 4F2 variants at pH 6.0 (100 mM phosphate, 200 mM NaCl) and pH 5.0 (100 mM sodium acetate, 200 mM NaCl) using DSC. Because the imidazole ring of histidine has a pKa value of 6.0 and below this pKa value, the protonated state of histidine residues can be alternated. Table I shows the Tm vale differences of the 4F2 variants at pH 6.0 and pH 5.0 from the pH 7.0 Tm values. At pH 5.0 conditions, the largest drop of Tm values was observed in all the 4F2 variants. VHHZnOBP1 shows the largest Tm decrease among pH 5.0 data with the value of –4.5 °C while wildtype cAbBCII10 show the smallest decrease with the value of –1.2 °C. 4F2 show second large decrease with the value of –3.7 °C and VHHsZnOBP3 shows the third with –2.1 °C. These data indicate that CDR1 is more susceptible to histidine protonation than CDR3. We initially expected that CDR3 contains four histidine residues and these histidine residues are important for stability. However, the DSC results show that the destabilization of VHHsZnOBP3 at pH 5.0 is relatively small. The Tm data at different pH conditions suggest histidine residues in CDR1 may be more critical for the stability than that in CDR3. Table I. The Tm difference (°C) from pH 7.0 values of 4F2 variants pH Wildtype 4F2 VHHZnOBP1 VHHsZnOBP3 7.0 0.0 °C 0.0 °C 0.0 °C 0.0 °C 6.0 –0.3 °C –0.8 °C –1.2 °C –0.4 °C 5.0 –1.2 °C –3.7 °C –4.5 °C –2.1 °C pH Wildtype 4F2 VHHZnOBP1 VHHsZnOBP3 7.0 0.0 °C 0.0 °C 0.0 °C 0.0 °C 6.0 –0.3 °C –0.8 °C –1.2 °C –0.4 °C 5.0 –1.2 °C –3.7 °C –4.5 °C –2.1 °C Buffers, 100 mM PO4 and 200 mM NaCl (pH 7.0, pH6.0) or 100 mM sodium acetate and 200 mM NaCl (pH 5.0), was used. Table I. The Tm difference (°C) from pH 7.0 values of 4F2 variants pH Wildtype 4F2 VHHZnOBP1 VHHsZnOBP3 7.0 0.0 °C 0.0 °C 0.0 °C 0.0 °C 6.0 –0.3 °C –0.8 °C –1.2 °C –0.4 °C 5.0 –1.2 °C –3.7 °C –4.5 °C –2.1 °C pH Wildtype 4F2 VHHZnOBP1 VHHsZnOBP3 7.0 0.0 °C 0.0 °C 0.0 °C 0.0 °C 6.0 –0.3 °C –0.8 °C –1.2 °C –0.4 °C 5.0 –1.2 °C –3.7 °C –4.5 °C –2.1 °C Buffers, 100 mM PO4 and 200 mM NaCl (pH 7.0, pH6.0) or 100 mM sodium acetate and 200 mM NaCl (pH 5.0), was used. To evaluate the general stability tendency of CAnIGET clones, we further measured the stability of the another CAnIGET VHH clone, AuE32, which binds to gold surface (3). The initial CDR1 grafting of a gold binding peptide (GBP: LKAHLPPSRLPS) resulted in a mutant VHHGBP1 and further CDR3 randomization/selection on VHHGBP1 resulted in the AuE32 VHH clone (CDR3 sequence: RRVRGGHLLR) as shown in Fig. 1. VHHGBP1 and AuE32 show the dissociation constants 3 μM and 150 nM toward gold surface, respectively (3). Both clones were purified using the same purification step of the 4F2 variants (Fig. 4a). We then performed the stability measurements of VHHGBP1 and AuE32 using DSC (Fig. 4b). AuE32 shows the Tm value of 61.0 °C while VHHGBP1 shows the Tm value of 62.3 °C. The CDR1 grafting resulted in the drop of the Tm by 16.0 °C which is similar de-stabilization of VHHZnOBP1 (Tm drop of 16.5 °C). Thus, the initial peptide grafting on CDR1 in the CAnIGET procedure may generally cause the de-stabilization. On the other hand, AuE32 shows a similar Tm value with that of VHHGBP1 with a little Tm drop by 1.3 °C. This stability tendency for the CDR3 randomization/selection step contrasts with that for 4F2, which recovers the stability after the final step. Though the reason for the difference of the stability impact on CDR3 between 4F2 and AuE32 is not clear, one possible reason might be the affinity difference toward material surfaces. In the case of 4F2, the parental VHH (VHHZnOBP1) already has decent affinity toward ZnO surface (Kd: 176 nM), while the parental VHH of AuE32 (VHHGBP1) has relatively low affinity toward gold surface (Kd: 3 μM). Thus, the CDR3 randomization/selection step could be harsher for AuE32 to survive the punning step. We speculate that the stably expressed clone on the phage surface may be relatively easily selected for the case of 4F2 while the less stable clone may be selected for the case of AuE32 because few phage clones with the decent affinity and stability may exist in the library. Fig. 4 View largeDownload slide (a) SDS-PAGE analysis of VHHGBP1 (lane 2) and AuE32 (lane 3). The result for cAbBCII10 and marker are also shown (lane 1and lane M). (b) DSC experiments of AuE32 variants at pH 7.0. Grey solid line: AuE32, Dotted line: VHHGBP1, Black sold line: cAbBCII10. Fig. 4 View largeDownload slide (a) SDS-PAGE analysis of VHHGBP1 (lane 2) and AuE32 (lane 3). The result for cAbBCII10 and marker are also shown (lane 1and lane M). (b) DSC experiments of AuE32 variants at pH 7.0. Grey solid line: AuE32, Dotted line: VHHGBP1, Black sold line: cAbBCII10. It has been reported that the wild-type VHH, cAbBCII-10, is suitable scaffold for CDR grafting (10). The scaffold accepts various types of CDR combinations, including anti-Lysozyme, anti-GFP and anti-RNase A VHH antibodies without significant stability loss. This stable scaffold is important basis for CAnIGET because the initial CDR1 grafting may reduce the stability and the scaffold must resist this initial de-stabilization. In summary, our results showing here demonstrate that the stability of VHH clones are drastically changed during the CAnIGET procedure (6). The initial peptide grafting at CDR1 leads to the de-stabilization of the VHH while subsequent randomization and selection steps on CDR3 show improvement of the stability for the 4F2 case while the case for AuE32 shows a little de-stabilization. To reveal the detailed mechanism of the stability change, further structural characterizations, such as X-ray crystallography, will be needed in the future study. Acknowledgements The authors thank Mrs. Daisuke Takeda and Ryosuke Sasaki for their technical assistances. This study was supported by JSPS KAKENHI Grant Numbers 24000011 and 26670051. Conflict of Interest None declared. References 1 Mirkin C.A. , Taton T.A. ( 2000 ) Materials chemistry: semiconductors meet biology . Nature 405 , 626 – 627 Google Scholar CrossRef Search ADS PubMed 2 Dickerson M.B. , Sandhage K.H. , Naik R.R. ( 2008 ) Protein- and peptide-directed syntheses of inorganic materials . Chem. Rev . 108 , 4935 – 4978 Google Scholar CrossRef Search ADS PubMed 3 Hattori T. , Umetsu M. , Nakanishi T. , Sawai S. , Kikuchi S. , Asano R. , Kumagai I. ( 2012 ) A high-affinity gold-binding camel antibody: antibody engineering for one-pot functionalization of gold nanoparticles as biointerface molecules . Bioconjugate Chem. 23 , 1934 – 1944 Google Scholar CrossRef Search ADS 4 Watanabe H. , Nakanishi T. , Umetsu M. , Kumagai I. ( 2008 ) Human anti-gold antibodies: biofunctionalization of gold nanoparticles and surfaces with anti-gold antibodies . J. Biol. 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Biol . 352 , 597 – 607 Google Scholar CrossRef Search ADS PubMed 11 Conrath K.E. , Lauwereys M. , Galleni M. , Matagne A. , Frère J.-M. , Kinne J. , Wyns L. , Muyldermans S. ( 2001 ) β-Lactamase inhibitors derived from single-domain antibody fragments elicited in the camelidae . Antimicrob. Agents Chemotherapy . 45 , 2807 – 2812 Google Scholar CrossRef Search ADS 12 Vincke C. , Loris R. , Saerens D. , Martinez-Rodriguez S. , Muyldermans S. , Conrath K. ( 2009 ) General strategy to humanize a camelid single-domain antibody and identification of a universal humanized nanobody scaffold . J. Biol. Chem . 284 , 3273 – 3284 Google Scholar CrossRef Search ADS PubMed 13 Umetsu M. , Mizuta M. , Tsumoto K. , Ohara S. , Takami S. , Watanabe H. , Kumagai I. , Adschiri T. ( 2005 ) Bioassisted room-temperature immobilization and mineralization of zinc oxide—the structural ordering of ZnO nanoparticles into a flower-type morphology . Adv. Mater. 17 , 2571 – 2575 Google Scholar CrossRef Search ADS Abbreviations Abbreviations CAnIGET construction of antibody by integrating grafting and evolution technology CDR complementarity determining region DSC differential scanning calorimetry SDS-PAGE sodium dodecyl sulfate poly acrylamide gel electrophoresis VHH VH of heavy chain antibody © The Author(s) 2018. Published by Oxford University Press on behalf of the Japanese Biochemical Society. All rights reserved This article is published and distributed under the terms of the Oxford University Press, Standard Journals Publication Model (https://academic.oup.com/journals/pages/about_us/legal/notices)

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The Journal of BiochemistryOxford University Press

Published: Jan 22, 2018

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